Use of CD24 for lowering low-density lipoprotein cholesterol levels

ABSTRACT

The present invention relates to the use of a CD24 protein for lowering low-density lipoprotein cholesterol levels, treating and preventing atherosclerosis, and for reducing risk of cardiovascular disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the national stage of International Application No.PCT/US2016/031109 filed May 6, 2016, which claims the benefit of U.S.Provisional Patent Application No. 62/158,157 filed May 7, 2015.

FIELD OF THE INVENTION

The present invention relates to the use of a CD24 protein for loweringlow-density lipoprotein cholesterol levels.

REFERENCE TO THE SEQUENCE LISTING

Applicant hereby makes reference to the sequence listing that iscontained in the file named “060275-0500-01USPC-Sequence-Listing.txt”(19 KB; created on Nov. 2, 2017), the contents of which are herebyincorporated herein by reference.

BACKGROUND OF THE INVENTION

Dyslipidemia, including high levels of low-density lipoprotein (LDL)cholesterol (LDL-C), is a major risk factor for atheroscleroticcardiovascular disease (ASCVD), which is the leading cause of death andmajor health care costs worldwide. The association between LDL-C and therisk of ASCVD has been established over decades of genetic andbiochemical studies, observational epidemiological and ecologicalstudies, and in vitro and animal experiments. Lowering LDL-C reducesASCVD events, demonstrating that LDL-C has a central, causal role incoronary heart disease (CHD) and ASCVD. Accordingly, fixed doses ofcholesterol-lowering drugs are used as the primary approach for reducingASCVD risk. In particular, elevated LDL-C is most frequently treatedwith statins (3-hydroxy-3-methylglutaryl-co-enzyme-A reductaseinhibitors). Statins lower LDL-C up to 50% from baseline, and reduceASCVD risk by 15-37%.

Yet, a residual 60-80% of ASCVD risk remains, which causes majorvascular events in about 20% of patients with CHD, even under optimalstatin treatment. Moreover, statins can cause muscle symptoms such aspain, tenderness, stiffness, cramping, weakness, fatigue, myopathy, andrhabdomyolysis. Further, although non-statin agents including bileacid-binding resins, fibrates, niacin, and ezetimibe significantlyimprove lipid profiles, none provides an additional risk reduction forcardiovascular events when combined with a statin. Accordingly, there isa need for improved non-statin drugs for lowering LDL-C levels andreducing the risk of ASCVD.

SUMMARY OF THE INVENTION

Provided herein is a method for reducing LDL-C levels in a subject byadministering a CD24 protein to a subject in need thereof. Also providedis a method for treating or preventing atherosclerosis in a subject byadministering the CD24 protein to a subject in need thereof. Furtherprovided is a method for lowering the risk of cardiovascular disease,which may be atherosclerotic cardiovascular disease, by administeringthe CD24 protein to a subject in need thereof. The subject may have anelevated LDL-C, which may be greater than or equal to 70, 75, or 190mg/dL. The subject may have been previously treated with anotherLDL-C-lowering drug, which may be a statin or an antagonist of PCSK9.

The CD24 protein may comprise the sequence of a mature human CD24 or avariant thereof. The mature human CD24 may comprise the sequence of SEQID NO: 1 or 2. The CD24 protein may comprise any or all of theextracellular domain of human CD24. The CD24 protein may comprise asignal sequence, which may allow secretion from a cell expressing theprotein. The signal peptide sequence may comprise the signal peptide ofhuman CD24, which may have SEQ ID NO: 4, or may be one that is found onother transmembrane or secreted proteins, or one modified from theexisting signal peptides known in the art. The CD24 protein may besoluble and may be glycosylated. The CD24 protein may be produced usinga eukaryotic protein expression system, which may comprise a vectorcontained in a Chinese Hamster Ovary cell line or areplication-defective retroviral vector. The replication defectiveretroviral vector may be stably integrated into the genome of aeukaryotic cell.

The CD24 protein may comprise a protein tag, which may be fused at theN- or C-terminus of the CD24 protein. The CD24 protein may comprise aportion of a mammalian immunoglobulin (Ig) protein, which may be human.The portion of the Ig protein may be a Fc region. The Fc region maycomprise the hinge region and CH2 and CH3 domains of the Ig protein andthe Ig protein may be IgG1, IgG2, IgG3, IgG4, or IgA. The Fc region mayalso comprise the hinge region and CH2, CH3, and CH4 domains of IgM. TheCD24 protein may comprise the sequence of SEQ ID NO: 5, 6, 8, 9, 11, or12.

Also provided herein is a method of monitoring the activity of the CD24protein in a subject. The method may comprise comparing the amount ofLDL-C in a blood sample obtained from the subject at a time point afteradministering the CD24 protein to the subject to an amount of LDL-C in ablood sample obtained from the subject before the time point. A decreasein the amount of LDL-C over time may be indicative of an increase inCD24 protein activity. The method may further comprise measuring theamount of LDL-C in the blood sample obtained after administering theCD24 protein to the subject. The method may also comprise measuring theamount of LDL-C in the blood sample obtained from the subject before thetime point. The amount of the CD24 protein subsequently administered tothe subject may be adjusted according to the amount of LDL-C in thesample obtained after administering the CD24 protein to the subject. Theamount of the CD24 protein in the subsequent administration may beadjusted to maintain a specific activity of concentration level of theCD24 protein in the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the amino acid composition of the full length CD24 fusionprotein, CD24Fc (also referred to herein as CD24Ig) (SEQ ID NO: 5). Theunderlined 26 amino acids are the signal peptide of CD24 (SEQ ID NO: 4),which are cleaved off during secretion from a cell expressing theprotein and thus missing from the processed version of the protein (SEQID NO: 6). The bold portion of the sequence is the extracellular domainof the mature CD24 protein used in the fusion protein (SEQ ID NO: 2).The last amino acid (A or V) that is ordinarily present in the matureCD24 protein has been deleted from the construct to avoidimmunogenicity. The non-underlined, non-bold letters are the sequence ofIgG1 Fc, including the hinge region and CH2 and CH3 domains (SEQ ID NO:7). FIG. 1B shows the sequence of CD24^(V)Fc (SEQ ID NO: 8), in whichthe mature human CD24 protein (bold) is the valine polymorphic variantof SEQ ID NO: 1. FIG. 1C shows the sequence of CD24^(A)Fc (SEQ ID NO:9), in which the mature human CD24 protein (bold) is the alaninepolymorphic variant of SEQ ID NO: 1. The various parts of the fusionprotein in FIGS. 1B and 1C are marked as in FIG. 1A and the variantvaline/alanine amino acid is double underlined.

FIG. 2 shows a method for purifying and processing a CD24 proteinexpressed from mammalian cell lines.

FIG. 3 shows amino acid sequence variations between mature CD24 proteinsfrom mouse (SEQ ID NO: 3) and human (SEQ ID NO: 1). The potentialO-glycosylation sites are bolded, and the N-glycosylation sites areunderlined.

FIG. 4A-C show WinNonlin compartmental modeling analysis ofpharmacokinetics of CD24Fc (CD24Ig) in mice. The opened circlesrepresent the average of 3 mice, and the line is the predictedpharmacokinetic curve. FIG. 4A. i.v. injection of 1 mg CD24Fc. FIG. 4B.s.c. injection of 1 mg CD24Fc. FIG. 4C. Comparison of the total amountsof antibody in the blood as measured by areas under curve (AUC),half-life and maximal blood concentration. Note that overall, the AUCand Cmax of the s.c. injection is about 80% of i.v. injection, althoughthe difference is not statistically significant.

FIG. 5 shows a plot of mean plasma CD24Fc concentration (±SD) bytreatment for a PK Evaluable Population in human subjects.PK=pharmacokinetic; SD=standard deviation.

FIG. 6 shows a dose proportionality plot of CD24Fc C_(max) versus dosefor a PK Evaluable Population.

FIG. 7 shows a dose proportionality plot of CD24Fc AUC_(0-42d) versusdose for a PK Evaluable Population.

FIG. 8 shows a dose proportionality plot of CD24Fc AUC_(0-inf) versusdose for a PK Evaluable Population.

DETAILED DESCRIPTION

The inventors have found that, surprisingly, proteins containing amature CD24 sequence are effective for lowering LDL-C levels, and areadditionally useful for treating and/or preventing atherosclerosis, andfor reducing the risk of cardiovascular disease such as atheroscleroticcardiovascular disease. As described in more detail herein, CD24 is asmall glycosyl-phosphatidyl-inositol (GPI)-anchored glycoprotein withwidespread expression among both hematopoietic and non-hematopoieticcells, which is encoded by a coding sequence of 240 base pairs. Of the80 amino acids, the first 26 constitute the signal peptide, while thelast 23 serve as a signal for cleavage to allow for the attachment ofthe GPI tail. As a result, the mature human CD24 molecule has only 31amino acids. Amino acid 31 is polymorphic among the human population. AC to T transition at nucleotide 226 results in the substitution ofalanine (A) with valine (V). Since this residue is in the positionimmediately N-terminal to the cleavage site, and since the replacementis non-conservative, these two alleles may be expressed at differentefficiencies on the cell surface. Transfection studies with copy DNAdemonstrated that CD24^(v/v) alleles are more efficiently expressed onthe cell surface. Consistent with this, CD24^(v/v) peripheral bloodleukocytes expressed higher levels of CD24, especially on T cells. Threelines of evidence demonstrate that CD24 is a genetic modifier for MS. Atthe population level, the CD24^(v/v) genotype is more than twice asfrequent as it is in the normal population. Among multiplex MS families,the CD24^(v) allele is preferentially transmitted to the MS patients incomparison to healthy controls. Furthermore, among the MS patients whohave a more severe form of the disease (Expanded Disability Status Scale[EDSS] at or exceeding 6.0, when the patients lose the ability to walkindependently), CD24^(v/v) individuals took, on average, 7 years toreach EDSS 6.0 from the first clinical symptom, yet the CD24^(a/v) orCD24^(a/a) individuals reached EDSS 6.0 in 13 to 15 years. Conversely, adinucleotide deletion in the 3′ untranslated region of CD24 messengerribonucleic acid (mRNA), which reduces CD24 mRNA stability and thusreduces CD24 expression, protects humans against MS and other autoimmunediseases. To date, CD24 has not been shown to affect lipid levels.

1. Definitions

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting. As used in thespecification and the appended claims, the singular forms “a,” “an” and“the” include plural referents unless the context clearly dictatesotherwise.

For recitation of numeric ranges herein, each intervening number therebetween with the same degree of precision is explicitly contemplated.For example, for the range of 6-9, the numbers 7 and 8 are contemplatedin addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1,6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitlycontemplated.

A “peptide” or “polypeptide” is a linked sequence of amino acids and maybe natural, synthetic, or a modification or combination of natural andsynthetic.

“Substantially identical” may mean that a first and second amino acidsequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% over a region of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150,160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or300 amino acids.

“Treatment” or “treating,” when referring to protection of an animalfrom a disease, means preventing, suppressing, repressing, or completelyeliminating the disease. Preventing the disease involves administering acomposition of the present invention to an animal prior to onset of thedisease. Suppressing the disease involves administering a composition ofthe present invention to an animal after induction of the disease butbefore its clinical appearance. Repressing the disease involvesadministering a composition of the present invention to an animal afterclinical appearance of the disease.

A “variant” may mean a peptide or polypeptide that differs in amino acidsequence by the insertion, deletion, or conservative substitution ofamino acids, but retain at least one biological activity. Representativeexamples of “biological activity” include the ability to bind to atoll-like receptor and to be bound by a specific antibody. Variant mayalso mean a protein with an amino acid sequence that is substantiallyidentical to a referenced protein with an amino acid sequence thatretains at least one biological activity. A conservative substitution ofan amino acid, i.e., replacing an amino acid with a different amino acidof similar properties (e.g., hydrophilicity, degree and distribution ofcharged regions) is recognized in the art as typically involving a minorchange. These minor changes can be identified, in part, by consideringthe hydropathic index of amino acids, as understood in the art. Kyte etal., J. Mol. Biol. 157:105-132 (1982). The hydropathic index of an aminoacid is based on a consideration of its hydrophobicity and charge. It isknown in the art that amino acids of similar hydropathic indexes can besubstituted and still retain protein function. In one aspect, aminoacids having hydropathic indexes of ±2 are substituted. Thehydrophilicity of amino acids can also be used to reveal substitutionsthat would result in proteins retaining biological function. Aconsideration of the hydrophilicity of amino acids in the context of apeptide permits calculation of the greatest local average hydrophilicityof that peptide, a useful measure that has been reported to correlatewell with antigenicity and immunogenicity. U.S. Pat. No. 4,554,101,incorporated fully herein by reference. Substitution of amino acidshaving similar hydrophilicity values can result in peptides retainingbiological activity, for example immunogenicity, as is understood in theart. Substitutions may be performed with amino acids havinghydrophilicity values within ±2 of each other. Both the hyrophobicityindex and the hydrophilicity value of amino acids are influenced by theparticular side chain of that amino acid. Consistent with thatobservation, amino acid substitutions that are compatible withbiological function are understood to depend on the relative similarityof the amino acids, and particularly the side chains of those aminoacids, as revealed by the hydrophobicity, hydrophilicity, charge, size,and other properties.

2. CD24

Provided herein is a CD24 protein, which may comprise the amino acidsequence of mature human CD24 or those from other mammals, whichcorresponds to the extracellular domain (ECD) of CD24, or a variantthereof. As described above, the sequence of the mature human CD24protein is 31 amino acids long with a variable alanine (A) with valine(V) residue at its C-terminal end:

SETTTGTSSNSSQSTSNSGLAPNPTNATTK(V/A) (SEQ ID NO: 1)

The C-terminal valine or alanine may be immunogenic and may be omittedfrom the CD24 protein to reduce its immunogenicity. Therefore, the CD24protein may comprise the amino acid sequence or mature human CD24lacking the C-terminal amino acid:

SETTTGTSSNSSQSTSNSGLAPNPTNATTK (SEQ ID NO: 2)

Despite considerable sequence variations in the amino acid sequence ofthe mature CD24 proteins from mouse and human, they are functionallyequivalent, as human CD24Fc has been shown to be active in the mouse.The amino acid sequence of the human CD24 ECD shows some sequenceconservation with the mouse protein (39% identity; Genbank accessionnumber NP_033976), as shown in FIG. 3. However, it is not thatsurprising that the percent identity is not higher as the CD24 ECD isonly 27-31 amino acids in length, depending on the species, and bindingto some of its receptor(s), such as Siglec 10/G, is mediated by itssialic acid and/or galactose sugars of the glycoprotein. The amino acidsequence identity between the extracellular domains of the humanSiglec-10 (GenBank accession number AF310233) and its murine homologSiglec-G (GenBank accession number NP_766488) receptor proteins is 63%.As a result of sequence conservation between mouse and human CD24primarily in the C-terminus and in the abundance of glycosylation sites,significant variations in the mature CD24 proteins may be tolerated inusing the CD24 protein, especially if those variations do not affect theconserved residues in the C-terminus or do not affect the glycosylationsites from either mouse or human CD24. Therefore, the CD24 protein maycomprise the amino acid sequence of mature murine CD24:

NQTSVAPFPGNQNISASPNPTNATTRG (SEQ ID NO: 3).

The amino acid sequence of the human CD24 ECD shows more sequenceconservation with the cynomolgus monkey protein (52% identity; UniProtaccession number UniProtKB-I7GKK1) than with mouse. Again, this is notsurprising given that the percent identity is not higher as the ECD isonly 29-31 amino acids in length in these species, and the role of sugarresidues in binding to its receptor(s). The amino acid sequence ofcynomolgous Siglec-10 receptor has not been determined but the aminoacid sequence identity between the human and rhesus monkey Siglec-10(GenBank accession number XP_001116352) proteins is 89%. Therefore, theCD24 protein may also comprise the amino acid sequence of maturecynomolgous (or rhesus) monkey CD24:

TVTTSAPLSSNSPQNTSTTPNPANTTTKA (SEQ ID NO: 10)

The CD24 protein may be soluble. The CD24 protein may comprise anN-terminal signal peptide, to allow secretion from a cell expressing theprotein. The signal peptide sequence may comprise the amino acidsequence MGRAMVARLGLGLLLLALLLPTQIYS (SEQ ID NO: 4). Alternatively, thesignal sequence may be any of those that are found on othertransmembrane or secreted proteins, or those modified from the existingsignal peptides known in the art.

a. Fusion

The CD24 protein may be fused at its N- or C-terminal end to a proteintag. The protein tag may comprise a portion of a mammalian Ig protein,which may be human or mouse or another species. The portion may comprisean Fc region of the Ig protein. The Fc region may comprise at least oneof the hinge region, CH2, CH3, and CH4 domains of the Ig protein. The Igprotein may be human IgG1, IgG2, IgG3, IgG4, or IgA, and the Fc regionmay comprise the hinge region, and CH2 and CH3 domains of the Ig. The Fcregion may comprise the human immunoglobulin G1 (IgG1) isotype, whichmay have the sequence of SEQ ID NO: 7. The Ig protein may also be IgM,and the Fc region may comprise the hinge region and CH2, CH3, and CH4domains of IgM. The protein tag may be an affinity tag that aids in thepurification of the protein, or a solubility-enhancing tag that enhancesthe solubility and recovery of functional proteins. The protein tag mayalso increase the valency of the CD24 protein. The protein tag may alsocomprise GST, His, FLAG, Myc, MBP, NusA, thioredoxin (TRX), smallubiquitin-like modifier (SUMO), ubiquitin (Ub), albumin, or a CamelidIg. Methods for making fusion proteins and purifying fusion proteins arewell known in the art.

Based on preclinical research, for the construction of the fusionprotein CD24Fc identified in the examples, the truncated form of nativeCD24 molecule of 30 amino acids, which lacks the final polymorphic aminoacid before the GPI signal cleavage site (that is, a mature CD24 proteinhaving SEQ ID NO: 2), has been used. The mature human CD24 sequence isfused to a human IgG1 Fc domain (SEQ ID NO: 7). The full length CD24Fcfusion protein is provided in SEQ ID NO: 5 (FIG. 1), and the processedversion of CD24Fc fusion protein that is secreted from the cell (i.e.lacking the signal sequence which is cleaved off) is provided in SEQ IDNO: 6. Processed polymorphic variants of mature CD24 (that is, matureCD24 protein having SEQ ID NO: 1) fused to IgG1 Fc may comprise SEQ IDNO: 11 or 12.

b. Production

The CD24 protein may be heavily glycosylated, and may be involved infunctions of CD24 such as costimulation of immune cells and interactionwith a damage-associated molecular pattern molecule (DAMP). The CD24protein may be prepared using a eukaryotic expression system. Theexpression system may entail expression from a vector in mammaliancells, such as Chinese Hamster Ovary (CHO) cells. The system may also bea viral vector, such as a replication-defective retroviral vector thatmay be used to infect eukaryotic cells. The CD24 protein may also beproduced from a stable cell line that expresses the CD24 protein from avector or a portion of a vector that has been integrated into thecellular genome. The stable cell line may express the CD24 protein froman integrated replication-defective retroviral vector. The expressionsystem may be GPEx™.

c. Pharmaceutical Composition

The CD24 protein may be contained in a pharmaceutical composition, whichmay comprise a pharmaceutically acceptable amount of the CD24 protein.The pharmaceutical composition may comprise a pharmaceuticallyacceptable carrier. The pharmaceutical composition may comprise asolvent, which may keep the CD24 protein stable over an extended period.The solvent may be PBS, which may keep the CD24 protein stable for atleast 66 months at −20° C. (−15˜−25° C.). The solvent may be capable ofaccommodating the CD24 protein in combination with another drug.

The pharmaceutical composition may be formulated for parenteraladministration including, but not limited to, by injection or continuousinfusion. Formulations for injection may be in the form of suspensions,solutions, or emulsions in oily or aqueous vehicles, and may containformulation agents including, but not limited to, suspending,stabilizing, and dispersing agents. The composition may also be providedin a powder form for reconstitution with a suitable vehicle including,but not limited to, sterile, pyrogen-free water.

The pharmaceutical composition may also be formulated as a depotpreparation, which may be administered by implantation or byintramuscular injection. The composition may be formulated with suitablepolymeric or hydrophobic materials (as an emulsion in an acceptable oil,for example), ion exchange resins, or as sparingly soluble derivatives(as a sparingly soluble salt, for example).

d. Dosage

The dose of the CD24 protein may ultimately be determined through aclinical trial to determine a dose with acceptable toxicity and clinicalefficacy. The initial clinical dose may be estimated throughpharmacokinetics and toxicity studies in rodents and non-human primates.The dose of the CD24 protein may be 0.01 mg/kg to 1000 mg/kg, and may be1 to 500 mg/kg, depending on the desired amount of LDL-C-lowering andthe route of administration. The CD24 protein may be administered byintravenous infusion or subcutaneous or intramural (that is, within thewall of a cavity or organ) injection, and the dose may be 10-1000 mg,10-500 mg, 10-240 mg, 10-120 mg, or 10, 30, 60, 120, or 240 mg, wherethe subject is a human.

3. Methods of Treatment

The CD24 protein may be administered to a subject to lower LDL-C levels,which may be elevated, and may further be elevated above a normal range.The normal range may be defined by a standard known in the art, such asNational Cholesterol Education Panel Adult Treatment Panel (ATP) III orthe 2005 update thereto (described in Circulation 2002; 106:3143-421;and J Am Coll Cardiol. 2004 Aug. 4; 44(3):720-32; the contents of bothwhich are incorporated herein by reference), or as set forth by theNational Institute for Health and Care Excellence (NICE). The subjectmay have a lysosomal acid lipase (LAL) deficiency, familialhypercholesterolemia, or hyperlipidemia. The CD24 protein may also beadministered to a subject to treat or prevent atherosclerosis, or tolower the risk of a cardiovascular disease event, which may be anatherosclerotic cardiovascular disease (ASCVD) event. The ASCVD eventmay be an acute coronary syndrome, myocardial infarction, stable orunstable angina, a coronary or other arterial revascularization, stroke,transient ischemic attack, or peripheral arterial disease presumed to beof atherosclerotic origin. The subject may be a mammal such as a human.

The subject may be a male or female. The subject may be of any age, butin particular may have an age of 40-75 years, or greater than 75 years.The subject may have a LDL-C greater than or equal to 70 mg/dL, 75mg/dL, or 190 mg/dL. The subject may also be diabetic or non-diabetic,be 40-75 years old, and have a LDL-C of 70-189 mg/dL. The subject mayhave a 10-year ASCVD risk (defined as nonfatal myocardial infarction,coronary heart disease death, or nonfatal and fatal stroke) greater thanor equal to 7.5%, or of 5-7.5%. The subject may have characteristics ofa subject for whom LDL-C lowering is recommended according to the 2013American College of Cardiology/American Heart Association Guidelines(Stone N J, et al., 2013 ACC/AHA guideline of the treatment of bloodcholesterol to reduce atherosclerotic cardiovascular risk in adults: areport of the American College of Cardiology/American Heart AssociationTask Force on Practice Guidelines, J Am Coll Cardiol 2014; 63:2889-934).The subject may have familial hypercholesterolemia, which may be causedby a mutation in the LDL receptor gene, apolipoprotein B gene, orpro-protein convertase subtilisin/kexintype 9 gene.

The subject may have been previously treated with a LDL-C-lowering drug,such as a statin. The subject may also have experienced an adverse eventas a result of the drug. The adverse event may have been a musclesymptom such as pain, tenderness, stiffness, cramping, weakness, orgeneral fatigue, and may have been a creatine phosphokinase levelindicative of an increased risk for adverse muscle events (which maybe >10 times the upper limit of normal). The subject may be recalcitrantto treatment with another cholesterol-lowering drug, and may have aLDL-C greater than or equal to 75 mg/dL after being treated with theother drug, which may be a statin. The subject may have graft vs. hostdisease, and may have exhibited a 10% or greater increase in LDL-C afterhaving undergone a transplant in comparison to the subject's LDL-Cbefore the transplant. The subject may have multiple sclerosis,rheumatoid arthritis, or an autoimmune or inflammatory disease.

a. Administration

The route of administration of the pharmaceutical composition may beparenteral. Parenteral administration includes, but is not limited to,intravenous, intraarterial, intraperitoneal, subcutaneous,intramuscular, intrathecal, intraarticular, and direct injection. Thepharmaceutical composition may be administered to a human patient, cat,dog, large animal, or an avian. The composition may be administered 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 times per day.

b. Combination Treatment

The CD24 protein may be combined with another treatment such as a drug,including a statin, a bile acid-binding resin, fibrate, niacin,ezetimibe, or a drug that increases LDL receptor levels, including butnot limited to an antibody or other inhibitor that antagonizes or blocksthe function of PCSK9. The CD24 protein and the other drug may beadministered together or sequentially.

The CD24 protein may be administered simultaneously or metronomicallywith other treatments. The term “simultaneous” or “simultaneously” asused herein, means that the CD24 protein and other treatment beadministered within 48 hours, preferably 24 hours, more preferably 12hours, yet more preferably 6 hours, and most preferably 3 hours or less,of each other. The term “metronomically” as used herein means theadministration of the agent at times different from the other treatmentand at a certain frequency relative to repeat administration.

The CD24 protein may be administered at any point prior to anothertreatment including about 120 hr, 118 hr, 116 hr, 114 hr, 112 hr, 110hr, 108 hr, 106 hr, 104 hr, 102 hr, 100 hr, 98 hr, 96 hr, 94 hr, 92 hr,90 hr, 88 hr, 86 hr, 84 hr, 82 hr, 80 hr, 78 hr, 76 hr, 74 hr, 72 hr, 70hr, 68 hr, 66 hr, 64 hr, 62 hr, 60 hr, 58 hr, 56 hr, 54 hr, 52 hr, 50hr, 48 hr, 46 hr, 44 hr, 42 hr, 40 hr, 38 hr, 36 hr, 34 hr, 32 hr, 30hr, 28 hr, 26 hr, 24 hr, 22 hr, 20 hr, 18 hr, 16 hr, 14 hr, 12 hr, 10hr, 8 hr, 6 hr, 4 hr, 3 hr, 2 hr, 1 hr, 55 mins., 50 mins., 45 mins., 40mins., 35 mins., 30 mins., 25 mins., 20 mins., 15 mins, 10 mins, 9 mins,8 mins, 7 mins., 6 mins., 5 mins., 4 mins., 3 mins, 2 mins, and 1 mins.The CD24 protein may be administered at any point prior to a secondtreatment of the CD24 protein including about 120 hr, 118 hr, 116 hr,114 hr, 112 hr, 110 hr, 108 hr, 106 hr, 104 hr, 102 hr, 100 hr, 98 hr,96 hr, 94 hr, 92 hr, 90 hr, 88 hr, 86 hr, 84 hr, 82 hr, 80 hr, 78 hr, 76hr, 74 hr, 72 hr, 70 hr, 68 hr, 66 hr, 64 hr, 62 hr, 60 hr, 58 hr, 56hr, 54 hr, 52 hr, 50 hr, 48 hr, 46 hr, 44 hr, 42 hr, 40 hr, 38 hr, 36hr, 34 hr, 32 hr, 30 hr, 28 hr, 26 hr, 24 hr, 22 hr, 20 hr, 18 hr, 16hr, 14 hr, 12 hr, 10 hr, 8 hr, 6 hr, 4 hr, 3 hr, 2 hr, 1 hr, 55 mins.,50 mins., 45 mins., 40 mins., 35 mins., 30 mins., 25 mins., 20 mins., 15mins., 10 mins., 9 mins., 8 mins., 7 mins., 6 mins., 5 mins., 4 mins., 3mins, 2 mins, and 1 mins.

The CD24 protein may be administered at any point after anothertreatment including about 1 min, 2 mins., 3 mins., 4 mins., 5 mins., 6mins., 7 mins., 8 mins., 9 mins., 10 mins., 15 mins., 20 mins., 25mins., 30 mins., 35 mins., 40 mins., 45 mins., 50 mins., 55 mins., 1 hr,2 hr, 3 hr, 4 hr, 6 hr, 8 hr, 10 hr, 12 hr, 14 hr, 16 hr, 18 hr, 20 hr,22 hr, 24 hr, 26 hr, 28 hr, 30 hr, 32 hr, 34 hr, 36 hr, 38 hr, 40 hr, 42hr, 44 hr, 46 hr, 48 hr, 50 hr, 52 hr, 54 hr, 56 hr, 58 hr, 60 hr, 62hr, 64 hr, 66 hr, 68 hr, 70 hr, 72 hr, 74 hr, 76 hr, 78 hr, 80 hr, 82hr, 84 hr, 86 hr, 88 hr, 90 hr, 92 hr, 94 hr, 96 hr, 98 hr, 100 hr, 102hr, 104 hr, 106 hr, 108 hr, 110 hr, 112 hr, 114 hr, 116 hr, 118 hr, and120 hr. The CD24 protein may be administered at any point prior after aprevious CD24 treatment including about 120 hr, 118 hr, 116 hr, 114 hr,112 hr, 110 hr, 108 hr, 106 hr, 104 hr, 102 hr, 100 hr, 98 hr, 96 hr, 94hr, 92 hr, 90 hr, 88 hr, 86 hr, 84 hr, 82 hr, 80 hr, 78 hr, 76 hr, 74hr, 72 hr, 70 hr, 68 hr, 66 hr, 64 hr, 62 hr, 60 hr, 58 hr, 56 hr, 54hr, 52 hr, 50 hr, 48 hr, 46 hr, 44 hr, 42 hr, 40 hr, 38 hr, 36 hr, 34hr, 32 hr, 30 hr, 28 hr, 26 hr, 24 hr, 22 hr, 20 hr, 18 hr, 16 hr, 14hr, 12 hr, 10 hr, 8 hr, 6 hr, 4 hr, 3 hr, 2 hr, 1 hr, 55 mins., 50mins., 45 mins., 40 mins., 35 mins., mins., 25 mins., 20 mins., 15mins., 10 mins., 9 mins., 8 mins., 7 mins., 6 mins., 5 mins., 4 mins., 3mins, 2 mins, and 1 mins.

4. Methods of Monitoring CD24 Protein Activity

The activity of the CD24 protein administered to a subject may bemonitored by detecting the concentration of LDL-C in the subject. Thesubject may be undergoing treatment with the CD24 protein, such astreatment for multiple sclerosis, rheumatoid arthritis,graft-versus-host disease, immune-mediated tissue injury, and the like.The concentration of LDL-C may be indicative of the level of CD24protein activity in the subject, where a decrease in LDL-C in thepatient indicates greater CD24 protein activity. The method may compriseobtaining a sample from the subject and detecting the amount of LDL-C inthe sample. The sample may be a blood sample such as serum or plasma.Methods of measuring LDL-C concentrations are well-known in the art,such as an ELISA based assay or a Colorimetric/Fluorometric assayfollowing cholesterol esterase and cholesterol oxidase treatment. Theamount of LDL-C may be measured by the Friedewald calculation, which maycomprise calculating the amount of LDL-C based on amounts of totalcholesterol, triglycerides, and high-density lipoprotein cholesterol(HDL-C) measured in the sample. The amount of HDL-C may be measuredeither by a precipitation procedure with dextran sulfate-Mg²⁺ or by adirect HDL-C assay. The amount of LDL-C may also be measured by theDIRECT LDL™ assay, the homogeneous N-GENEOUS™ LDL assay, or calculatedLDL-C values deriving from the ApoB based equation:0.41TC-0.32TG+1.70ApoB-0.27, (Clin Chem 1997; 43:808-815; the contentsof which are incorporated herein by reference). The level of LDL-C canbe monitored over time and during the course of CD24 protein treatmentin order to monitor the response to treatment.

The amount of CD24 protein being administered to the subject, fortreating an indication described herein or known in the art, may beadjusted based on the level of CD24 protein activity detected usingLDL-C. The level of LDL-C can be monitored over a period of time orduring the course of CD24 protein treatment. If the LDL-C concentrationin the subject is reduced to a level within the range of normal, thenthe amount of CD24 protein administered to the subject may be reduced,such as by lowering the dose of CD24 protein or administering it lessfrequently. If the LDL-C concentration remains unchanged or remainsabove the range of normal, then the amount of CD24 protein administeredto the subject may be increased, such as by increasing the dose of CD24protein or administering it more frequently. As an alternative to LDL-C,the concentration of LDL particles (LDL-P) may also be measured tomonitor CD24 protein activity. The LDL-P concentration may be detecteddirectly using NMR.

Levels of the CD24 protein administered to the subject may also bemonitored, which may be by a method comprising obtaining a sample fromthe subject and detecting the amount of the CD24 protein in the sample.The sample may be a blood sample such serum or plasma. Protein detectionmethods are well-known in the art. The CD24 protein in the sample may bedetected by any protein detection method, such as an immunoassayincluding ELISA, Gyros, MSD, Biacore, AlphaLISA, Delfia, Singulex,Luminex, Immuno-PCR, Cell-based assays, RIA, Western blot, an affinitycolumn, and the like. The ELISA method may be sandwich ELISA orcompetitive ELISA. For example, the ELISA may comprise contacting thesample to an anti-CD24 protein antibody, contacting the CD24protein-CD24 protein antibody complex with a labeled antibody that bindsto the anti-CD24 protein antibody, and measuring the amount of labeledantibody by detecting a signal produced by the label, where the amountof signal correlates to the amount of CD24 protein in the sample.

The amount of CD24 protein administered to the subject may be adjusted(such as by adjusting dose and frequency of administration) based on apharmacokinetic parameter for the CD24 protein. For example, the amountof CD24 protein administered to the subject may be adjusted to obtain aplasma CD24 concentration of greater than 1 ng/ml. In another example,the amount of CD24 protein administered to the subject is adjusted tomaintain a steady state plasma concentration greater than 1 ng/mL. Inanother example, the amount of CD24 protein administered to the subjectmay be adjusted to obtain a C_(max) of the CD24 protein of at leastabout 1 ng/mL. In yet another example, the amount of CD24 proteinadministered to the subject may be adjusted to achieve a drug exposurelevel, as defined by the AUC_(0-inf), of the CD24 protein of at leastabout 400,000 ng*hr/mL.

The present invention has multiple aspects, illustrated by the followingnon-limiting examples.

Example 1 Making Soluble CD24 Proteins

The mature sequence of CD24 was fused to IgG1 Fc. The amino acidcomposition of the CD24 fusion protein is provided in FIG. 1. Areplication-defective retroviral vector that drives expression of theCD24Fc fusion protein was then generated. The GPEx™ (an acronym for geneproduct expression) system offers several important advantages, the mostimportant of which is the, on average, >1000 insertions/cell but withonly 1 copy/insertion. Moreover, since the retrovirus preferentiallyinserts into the transcriptional active locus, the GPEx™ resulted in ahigh level of expression of the targeted protein. Stable cell lines thatproduce a high yield of CD24Fc were generated. In addition 45 grams ofGLP grade products and ˜100 grams of cGMP grade products were produced.The methods used for downstream processing of media harvested from thebioreactor are summarized in the flow chart below (FIG. 2).

Harvest Clarification

The bioreactor culture media was clarified using Cuno 60M02 Maximizerdepth filters followed by a Millipore Opticap 0.22 um filter. Thefiltrate was collected into a sterile collection bag. Samples wereobtained for CD24-Fc yield quantitation by ELISA.

Protein A Capture

The clarified media was passed over a column of Protein A resin (GEHealthcare MabSelect) at a concentration not exceeding 16 g/L of resin(based on ELISA) and a contact time of 4 minutes. The column was washedwith the equilibration buffer (50 mM Tris+0.15M NaCl pH7.5), then with10 mM sodium citrate/citric acid pH 6.0 for 5cvs. Bound CD24Fc waseluted from the column using 10 mM sodium citrate/citric acid pH 3.5

Viral Inactivation

The Protein A eluate fraction was immediately brought to pH 3.0 with theaddition of 2M Hydrochloric acid and held at this pH for 30 minutes atambient temperature. It was then brought to pH 5.0 with the addition of1M Tris base, and filtered to clarity using a 0.65 um glass fiber filter(Sartorius Sartopure GF2) and 0.2 um (Sartorius Sartopore 2) into asterile collection bag.

SP-Sepharose Chromatography

The viral inactivated material was applied to a column of SP-Sepharose(GE Healthcare) at a concentration not exceeding 25 g/L of resin (basedon A280 nm of 1.22=1 mg/mL) and a linear flow rate of 250 cm/hr. Thecolumn was washed with the equilibration buffer (10 mM sodiumcitrate/citric acid pH 5.0) and bound CD24Fc was eluted from the columnusing 10 mM sodium citrate/citric acid+0.2M NaCl pH5.0. The effluent wascollected into a sterile collection bag.

Mustang Q Chromatography

The SP-Sepharose elute was adjusted to pH 7.5 by the addition of 1M Trisbase and diluted with WFI to reduce the conductivity. The dilutedmaterial was applied to a Mustang Q filter (Pall) at a concentration notexceeding 0.5 g/L of resin (based on A280 nm of 1.22=1 mg/mL) and at aflow rate of 5 column volumes/minute. The filter was washed with theequilibration buffer (10 mM Tris pH 7.5) and the CD24-Fc is contained inthe flow through and is collected into a sterile collection bag.

Viral Filtration

The Mustang Q flow through was then filtered at a constant pressure of30 psi through a 0.2 mM filter and a Millipore NFP viral filter (nominalpore size 20 nm) and was collected into a sterile collection bag.

Concentration and Final Formulation

The product was concentrated and diafiltered using a 10 kDaultrafiltration membrane (Millipore Prep/Scale) into a 10 mM sodiumphosphate, 150 mM sodium chloride pH 7.2 at approximately 10 mg/mL finalconcentration as determined by absorbance at 280 nm. Analytical sampleswere drawn from the bulk whilst in a biosafety cabinet. Labeling wasperformed and the samples were delivered to QC for testing while thebulk aliquots were stored at 2-8° C. pending release.

Example 2 CD24 Pharmacokinetics in Mice

1 mg of CD24Fc (CD24Fc) was injected into naïve C57BL/6 mice andcollected blood samples at different timepoints (5 min, 1 hr, 4 hrs, 24hrs, 48 hrs, 7 days, 14 days and 21 days) with 3 mice in each timepoint.The sera were diluted 1:100 and the levels of CD24Fc was detected usinga sandwich ELISA using purified anti-human CD24 (3.3 μg/ml) as thecapturing antibody and peroxidase conjugated goat anti-human IgG Fc (5μg/ml) as the detecting antibodies. As shown in FIG. 4a . The decaycurve of CD24Fc revealed a typical biphase decay of the protein. Thefirst biodistribution phase had a half-life of 12.4 hours. The secondphase follows a model of first-order elimination from the centralcompartment. The half-life for the second phase was 9.54 days, which issimilar to that of antibodies in vivo. These data suggest that thefusion protein is very stable in the blood stream. In another study inwhich the fusion protein was injected subcutaneously, an almostidentical half-life of 9.52 days was observed (FIG. 4b ). Moreimportantly, while it took approximately 48 hours for the CD24Fc toreach peak levels in the blood, the total amount of the fusion proteinin the blood, as measured by AUC, was substantially the same by eitherroute of injection. Thus, from therapeutic point of view, differentroute of injection should not affect the therapeutic effect of the drug.This observation greatly simplified the experimental design for primatetoxicity and clinical trials.

Example 3 CD24 Lowers LDL-C Levels

This example demonstrates that CD24 lowers LDL-C. Changes of fastingLDL-C in plasma from baseline were analyzed in a clinical study which isdescribed in more detail below (see the Methods section of thisexample). Fasting LDL-C levels were determined among samples obtained onDay −1, Day 7, and Day 42 for Cohort 1 (CD24Fc 10 mg group). Beginningwith Cohort 2 (CD24Fc 30 mg group), this lipid sampling was expanded toinclude Day 14. The data are summarized in Table 1. Due to an incompletedataset in Cohort 1, Cohorts 2-5 were used to analyze for dose-dependentreduction of LDL-C levels. A statistically significant dose-dependentreduction was observed (Table 1).

TABLE 1 Change in LDL-C levels on Day 7 (U1), Day 14 (U2) and Day 42(U3) from baseline (U0, defined as 100%) Dose Obs Variable Label N MeanStd Dev Minimum Maximum 10 mg 6 u0 Baseline LDL 6 100.0000000 0100.0000000 100.0000000 u1  7 days LDL ratio 5 99.6785886 8.566550587.0370370 107.7586207 u2 14 days LDL ratio 0 . . . . u3 42 days LDLratio 6 102.9957054 5.3134796 96.8085106 110.5769231 30 mg 6 u0 BaselineLDL 6 100.0000000 0 100.0000000 100.0000000 u1  7 days LDL ratio 696.9190313 9.5257894 86.9047619 113.4328358 u2 14 days LDL ratio 697.5816504 15.2482354 84.5238095 122.3880597 u3 42 days LDL ratio 6106.1959745 8.2383407 95.2830189 113.4328358 60 mg 6 u0 Baseline LDL 6100.0000000 0 100.0000000 100.0000000 u1  7 days LDL ratio 6 90.762058812.6697467 72.0720721 106.1728395 u2 14 days LDL ratio 6 102.56711705.2461286 96.5517241 110.3773585 u3 42 days LDL ratio 6 105.154694313.4340830 93.2773109 127.1604938 120 mg  6 u0 Baseline LDL 6100.0000000 0 100.0000000 100.0000000 u1  7 days LDL ratio 6 87.147663216.0595374 61.7391304 106.4516129 u2 14 days LDL ratio 6 95.262541811.8341667 83.4782609 116.1290323 u3 42 days LDL ratio 6 100.13771659.9404474 87.1794872 112.3456790 240 mg  6 u0 Baseline LDL 6 100.00000000 100.0000000 100.0000000 u1*  7 days LDL ratio 6 84.6472221 7.655389671.5596330 94.0476190 u2* 14 days LDL ratio 5 90.1393086 5.250180786.2385321 99.0825688 u3 42 days LDL ratio 6 107.0369419 14.715479679.8449612 121.1009174 Control 10 u0 Baseline LDL 10 100.0000000 0100.0000000 100.0000000 u1  7 days LDL ratio 10 93.7350811 8.974712183.7837838 107.1428571 u2 14 days LDL ratio 8 104.5965396 13.862595283.7837838 125.2631579 u3 42 days LDL ratio 10 102.6699920 16.281559977.0270270 138.1578947 *P < 0.05 when compared to placebo group, studentt-test.

Using cohort 1 as reference, it was determined whether CD24Fc reducedLDL-C levels in a dose- and time-dependent manner. As shown in Table 2,compared with cohort 1 which received 10 mg of CD24Fc, a significantdose-dependent reduction of LDL-C levels was observed (p<0.0001).

TABLE 2 Dose and time-dependence of LDL-C reduction in Cohorts by GEEmodel, using cohort 1 (the lowest dose as reference) Standard 95%Confidence Parameter Estimate Error Limits Z Pr > |Z| Intercept 98.05445.4745 87.3245 108.7842 17.91 <.0001 time 1.6471 2.1861 −2.6375 5.93170.75 0.4512  30 mg 3.7167 7.3244 −10.6389 18.0722 0.51 0.6118 time * 30mg −1.4733 3.5435 −8.4183 5.4718 −0.42 0.6776  60 mg −25.4898 14.4124−53.7377 2.7581 −1.77 0.0770 time * 60 mg 10.7245 5.0225 0.8805 20.56852.14 0.0327 120 mg −21.2684 9.4771 −39.8431 −2.6936 −2.24 0.0248 time *120 mg 6.6669 3.9357 −1.0468 14.3806 1.69 0.0903 240 mg −15.8681 6.9247−29.4402 −2.2960 −2.29 0.0219 time * 240 mg 5.4390 2.8825 −0.210611.0887 1.89 0.0592

A statistically significant dose-dependent reduction of LDL-C wasobserved, indicating that CD24Fc is effective for lowering LDL-C inhuman patients.

Methods

This was a Phase I, randomized, double-blind, placebo-controlled, singleascending dose study to assess the safety, tolerability, and PK ofCD24Fc in healthy male and female adult subjects. A total of 40 subjectsin 5 cohorts of 8 subjects each were enrolled in this study. Six of the8 subjects in each cohort received study drug and 2 subjects receivedplacebo (0.9% sodium chloride, saline). The first cohort was dosed with10 mg. Succeeding cohorts received 30 mg, 60 mg, 120 mg, and 240 mg ofCD24Fc or matching placebo and were dosed at least 3 weeks apart toallow for review of safety and tolerability data for each prior cohort.Administration of the next higher dose to a new cohort of subjects waspermitted only if adequate safety and tolerability had beendemonstrated.

In each cohort, the initial 2 subjects were 1 study drug recipient and 1placebo recipient on Day 1. The 3rd to 5th and 6th to 8th subjects weredosed after Day 7 (a minimum of 24 hours apart between the subgroups).Each subject was dosed at least 1 hour apart in the same subgroup. Ifnecessary, dosing of the rest of subjects was delayed pending review ofany significant safety issues that may have arisen during the post-doseperiod involving the first or second subgroups in that cohort. Thesubsequent cohort was dosed at least 3 weeks after the prior cohort.

Screening Period:

The Screening Visit (Visit 1) occurred up to 21 days prior to thebeginning of the active treatment period. After providing informedconsent, subjects underwent screening procedures for eligibility.

Treatment Period:

Subjects were admitted to the Clinical Pharmacology Unit (CPU) on Day −1(Visit 2), and the randomized treatment period began on Day 1 followinga 10-hour minimum overnight fast. Subjects were randomly assigned totreatment with CD24Fc or placebo as a single dose. Subjects remainedconfined until the morning of Day 4.

Follow-Up:

All subjects returned to the CPU on Day 7, Day 14, Day 21, Day 28, andDay 42 (±1 day) for follow-up visits (Visit 3, Visit 4, Visit 5, Visit6, and Visit 7). Visit 7 was the final visit for all subjects.

Duration of Treatment:

The total study duration for each subject was up to 63 days. Single-doseadministration occurred on Day 1.

Number of Subjects:

Planned: 40 subjects

Screened: 224 subjects

Randomized: 40 subjects

Completed: 39 subjects

Discontinued: 1 subject

Diagnosis and Main Criteria for Inclusion:

The population for this study was healthy males and females between theages of 18 and 55 years, inclusive, with a body mass index between 18kg/m² and 30 kg/m², inclusive.

Investigational Product and Comparator Information:

CD24Fc: single dose of 10 mg, 30 mg, 60 mg, 120 mg, or 240 mgadministered via IV infusion; lot number: 09MM-036. CD24Fc was a fullyhumanized fusion protein consisting of the mature sequence of human CD24and the fragment crystallizable region of human immunoglobulin G1(IgG1Fc). CD24Fc was supplied as a sterile, clear, colorless,preservative-free, aqueous solution for IV administration. CD24Fc wasformulated as single dose injection solution, at a concentration of 10mg/mL and a pH of 7.2. Each CD24Fc vial contained 160 mg of CD24Fc, 5.3mg of sodium chloride, 32.6 mg of sodium phosphate dibasic heptahydrate,and 140 mg of sodium phosphate monobasic monohydrate in 16 mL±0.2 mL ofCD24Fc. CD24Fc was supplied in clear borosilicate glass vials withchlorobutyl rubber stoppers and aluminum flip-off seals.

Matching placebo (0.9% sodium chloride, saline) administered via IVinfusion; lot numbers: P296855, P311852, P300715, P315952.

The intent-to-treat (ITT) Population consisted of all subjects whoreceived at least 1 dose of the study drug. The ITT Population was theprimary analysis population for subject information and safetyevaluation.

Clinical laboratory evaluations (chemistry, hematology, and urinalysis)were summarized by treatment and visit. Change from baseline was alsosummarized. Vital signs (blood pressure, heart rate, respiratory rate,and temperature) were summarized by treatment and time point. Changefrom baseline was also summarized. All physical examination data werelisted. Electrocardiogram parameters and the change from baseline weresummarized. Overall interpretations were listed. Fasting LDL-C and highdensity lipoprotein cholesterol were obtained on Day −1, Day 7, and Day42 for Cohort 1 (CD24Fc 10 mg group). Beginning with Cohort 2 (Cd24Fc 30mg group), this lipid sampling was expanded to include Day 14.

Example 4 CD24 Pharmacokinetics in Humans

This example shows an analysis of the pharmacokinetics of a CD24 proteinin humans.

Plasma CD24Fc Concentration

As shown in FIG. 5, the mean plasma concentration of CD24Fc increasedproportionally to the dose of CD24Fc administered. For all dose groupsexcept 120 mg, the maximum mean plasma concentration of CD24Fc wasreached at 1 hour post-dose. The maximum mean plasma concentration ofCD24Fc for the 120 mg group was reached at 2 hours post-dose. By Day 42(984 hours), the mean plasma concentration of CD24Fc for all groups haddecreased to between 2% and 4% of the maximum mean plasma concentration.

Table 3 summarizes the plasma CD24Fc PK parameters by treatment for thePK Evaluable Population.

TABLE 3 Summary of Plasma CD24Fc Pharmacokinetic Parameters byTreatment - PK Evaluable Population CD24Fc CD24Fc CD24Fc CD24Fc CD24FcParameter 10 mg 30 mg 60 mg 120 mg 240 mg Statistic (N = 6) (N = 6) (N =6) (N = 6) (N = 6) C_(max) (ng/mL) n 6 6 6  6  6  Mean (SD) 2495 (576) 9735 (1715) 30 083 (7179)   52 435 (9910)   95 865 (10 734) CV % 23.117.6 23.9 18.9 11.2 Median 2371 9218 29 026    50 401    93 206    Min,Max 1,967, 8,583, 22,557, 40,434, 81,296, 3,390 13,086 42,628 65,704110,110 Geometric mean 2,442 9,625 29,424    51,666    95,365   Geometric CV % 22.8 16.1 23.0 19.0 11.2 AUC_(0-42 d) (ng * hr/mL) n 6 66  6  6  Mean (SD) 423,061 (99,615)  1,282,430 (88,798)   3,226,255(702,862)   6,541,501 (2,190,944) 12,704,705 (1,918,596)  CV % 23.5 6.921.8 33.5 15.1 Median 434,043 1,302,719 3,124,933      5,785,142     12,563,426      Min, Max 291,020, 1,175,733, 2,487,550, 4,485,193,10,466,635, 528,079 1,403,024 4,139,748 9,415,266 15,693,606 Geometricmean 412,795 1,279,851 3,163,252      6,249,552      12,586,731     Geometric CV % 25.0 7.0 22.0 33.8 15.0 AUC_(0-inf) (ng * hr/mL) n 6 6 6 6  6  Mean (SD) 462,260 (116,040) 1,434,464 (131,316)   3,497,196(705,653)   7,198,196 (2,458,320) 13,861,796 (1,962,780)  CV % 25.1 9.220.2 34.2 14.2 Median 470,426 1,422,205 3,519,732      6,463,665     13,713,034      Min, Max 310,956, 1,281,715, 2,703,655, 4,910,640,11,822,988, 596,599 1,650,503 4,309,023 10,479,940 17,175,236 Geometricmean 449,583 1,429,578 3,437,036      6,862,129      13,750,972     Geometric CV % 26.7 9.0 20.7 34.6 13.8 T_(max) (hr) n 6 6 6  6  6  Mean(SD) 1.15 (0.42) 1.17 (0.41) 1.01 (0.01) 1.34 (0.51) 1.33 (0.52) CV %36.1 35.0  1.2 38.0 38.7 Median 1.00 1.00  1.00  1.03  1.00 Min, Max0.92, 2.00 1.00, 2.00 1.00, 1.03 1.00, 2.00 1.00, 2.00 t½ (hr) n 6 6 6 6  6  Mean (SD) 280.83 (22.37)  327.10 (41.32)  279.82 (65.59)  286.45(23.38)  285.33 (24.33)  CV % 8.0 12.6 23.4  8.2  8.5 Median 279.61317.23 264.69 290.76 287.74 Min, Max 258.87, 321.26 289.82, 394.24210.18, 362.46 243.89, 309.26 249.24, 322.26 AUCextr (%) n 6 6 6  6  6 Mean (SD) 7.61 (2.14) 10.44 (2.94)  7.88 (4.26) 8.92 (1.94) 8.46 (1.99)CV % 28.1 28.2 54.0 21.8 23.5 Median 7.16 10.01  6.35  9.27  8.45 Min,Max  5.46, 11.47  7.10, 15.05  3.92, 14.48  5.49, 10.99  5.56, 11.50 CL(L/hr) n 6 6 6  6  6  Mean (SD) 0.0229 (0.0061) 0.0211 (0.0019) 0.0178(0.0036) 0.0183 (0.0058) 0.0176 (0.0023) CV % 26.7 8.8 20.5 31.7 13.3Median 0.0216 0.0211   0.0173   0.0191   0.0175 Min, Max 0.0168, 0.03220.0182, 0.0234 0.0139, 0.0222 0.0115, 0.0244 0.0140, 0.0203 Vd (L) n 6 66  6  6  Mean (SD) 9.153 (1.943) 9.867 (0.804) 7.289 (2.592) 7.491(2.202) 7.276 (1.426) CV % 21.2 8.1 35.6 29.4 19.6 Median 8.507 10.007  7.486   7.691   7.151 Min, Max 7.326, 12.010 8.771, 10.958  4.222,11.139 4.933, 9.974 5.814, 9.438 AUC_(0-42 d) = area under theconcentration-time curve from time 0 to 42 days; AUC_(0-inf) = areaunder the concentration-time curve extrapolated from time 0 to infinity;AUC_(extr) = percentage of AUC_(0-inf) that was due to extrapolationfrom the time of the last measurable concentration, per subject, toinfinity; CL = total body clearance; C_(max) = maximum observed plasmadrug concentration; CV % = coefficient of variation; Min = minimum; Max= maximum; SD = standard deviation; t 1/2 = terminal eliminationhalf-life; T_(max) = time of maximum observed plasma drug concentration;V_(d) = volume of distribution.

Plasma CD24Fc Dose Proportionality Analysis

FIG. 6 shows a dose proportionality plot of CD24Fc C_(max) versus dosefor the PK Evaluable Population. FIG. 7 shows a dose proportionalityplot of CD24Fc AUC_(0-42d) versus dose for the PK Evaluable Population.FIG. 8 shows a dose proportionality plot of CD24Fc AUC_(0-inf) versusdose for the PK Evaluable Population. Table 4 shows a power analysis ofdose proportionality.

TABLE 4 Power Analysis of Dose Proportionality: Plasma CD24FcPharmacokinetic Parameters - PK Evaluable Population CD24Fc CD24FcCD24Fc CD24Fc CD24Fc Dose Proportionality Parameter 10 mg 30 mg 60 mg120 mg 240 mg Slope Standard Statistic (N = 6) (N = 6) (N = 6) (N = 6)(N = 6) Estimate Error 90% CI C_(max) (ng/mL) 1.172 0.040 (1.105, 1.240)Geometric mean 2,441.8 9,624.9 29,424.4 51,666.4 95,364.9 Geometric CV %22.8 16.1 23.0 19.0 11.2 AUC_(0-42 d) 1.088 0.036 (1.027, 1.148) (ng *hr/mL) Geometric mean 412,794.8 1,279,850.8 3,163,251.7 6,249,551.912,586,731.3 Geometric CV % 25.0 7.0 22.0 33.8 15.0 AUC_(0-inf) (ng *hr/mL) 1.087 0.036 (1.026, 1.148) Geometric mean 449,583.5 1,429,577.53,437,035.6 6,862,128.7 13,750,972.4 Geometric CV % 26.7 9.0 20.7 34.613.8 Geometric CV % = 100 * sqrt(exp(SD²) − 1), where SD was thestandard deviation of the log-transformed data. The power model wasfitted by restricted maximum likelihood, regressing the log-transformedPK parameter on log transformed dose. Both the intercept and slope werefitted as fixed effects. Dose proportionality was not rejected if the90% CI lies within (0.8, 1.25). AUC_(0-42 d) = area under theconcentration-time curve from time 0 to 42 days; AUC_(0.inf) = areaunder the concentration-time curve extrapolated from time 0 to infinity;CI = confidence interval; C_(max) = maximum observed plasma drugconcentration; CV % = coefficient of variation; PK = pharmacokinetic; SD= standard deviation.

The C_(max) slope estimate was 1.172 with a 90% CI of 1.105 to 1.240.The AUC_(0-42d) slope estimate was 1.088 with a 90% CI of 1.027 to1.148. The AUC_(0-inf) slope estimate was 1.087 with a 90% CI of 1.026to 1.1.

Pharmacokinetic Conclusions

The C_(max) and AUCs of plasma CD24Fc increased proportionally to thedoses administered in mouse, monkey and human. The plasma CD24Fc reachedT_(max) between 1.01 and 1.34 hours. The t_(1/2) of plasma CD24Fc rangedbetween 280.83 and 327.10 hours.

The invention claimed is:
 1. A method of reducing low-densitylipoprotein (LDL) cholesterol (LDL-C) levels in a human subject in needthereof, comprising administering a CD24 protein to the subject, whereinthe CD24 protein comprises: (a) a mature human CD24 polypeptidecomprising the sequence set forth in SEQ ID NO: 1 or 2; and, (b) a Fcregion of a human IgG protein; and, wherein the Fc region is fused atthe C-terminus of the CD24 protein.
 2. The method of claim 1, whereinthe subject has an elevated LDL-C.
 3. The method of claim 2, wherein thesubject has a LDL-C greater than or equal to 75 mg/dL.
 4. The method ofclaim 2, wherein the subject has a LDL-C of greater than or equal to 70mg/dL or 190 mg/dL.
 5. The method of claim 1, wherein the subject hasbeen previously treated with another LDL-C-lowering drug.
 6. The methodof claim 5, wherein the other LDL-C-lowering drug is a statin.
 7. Themethod of claim 5, wherein the other LDL-C-lowering drug is anantagonist of PCSK9.
 8. The method of claim 1, wherein the CD24 proteinis soluble.
 9. The method of claim 1, wherein the CD24 protein isglycosylated.
 10. The method of claim 1, wherein the Fc region comprisesa hinge region and CH2 and CH3 domains of the human IgG protein.
 11. Themethod of claim 10, wherein the human IgG protein is selected from thegroup consisting of IgG1, IgG2, IgG3, and IgG4.
 12. The method of claim11, wherein the CD24 protein comprises the sequence set forth in SEQ IDNO: 6, ii, or
 12. 13. The method of claim 1, wherein the CD24 protein isproduced using a eukaryotic protein expression system.
 14. The method ofclaim 13, wherein the expression system comprises a vector contained ina Chinese Hamster Ovary cell line or a replication-defective retroviralvector.
 15. The method of claim 14, wherein the replication-defectiveretroviral vector is stably integrated into the genome of a eukaryoticcell.
 16. A method of treating atherosclerosis in a human subject inneed thereof, comprising administering a CD24 protein to the subject,wherein the amino acid sequence of the CD24 protein consists of SEQ IDNO: 6, 11, or
 12. 17. A method of lowering the risk of atheroscleroticcardiovascular disease in a human subject in need thereof, comprisingadministering a CD24 protein to the subject, wherein the amino acidsequence of the CD24 protein consists of SEQ ID NO: 6, 11, or
 12. 18.The method of claim 12, wherein the amino acid sequence of the CD24protein consists of the sequence set forth in SEQ ID NO: 6, ii or 12.19. The method of claim 16, wherein the CD24 protein is soluble.
 20. Themethod of claim 17, wherein the CD24 protein is soluble.
 21. A method ofreducing LDL-C levels in a human subject in need thereof, comprisingadministering to the subject a CD24 protein consisting of the sequenceset forth in SEQ ID NO: 6.